MEP is a consultant for Sucampo. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: ASC RCR CAK MEP. Performed the experiments: ASC. Analyzed the data: ASC CMF RCP SD. Contributed reagents/materials/analysis tools: MEP PY. Wrote the paper: ASC RCR CAK. Designed the software used in analysis: YW. Provided 5-HT1A knockout mice: RH.
To assess the neuroprotective effects of flibanserin (formerly BIMT-17), a dual 5-HT1A agonist and 5-HT2A antagonist, in a light-induced retinopathy model.
Albino BALB/c mice were injected intraperitoneally with either vehicle or increasing doses of flibanserin ranging from 0.75 to 15 mg/kg flibanserin. To assess 5-HT1A-mediated effects, BALB/c mice were injected with 10 mg/kg WAY 100635, a 5-HT1A antagonist, prior to 6 mg/kg flibanserin and 5-HT1A knockout mice were injected with 6 mg/kg flibanserin. Injections were administered once immediately prior to light exposure or over the course of five days. Light exposure lasted for one hour at an intensity of 10,000 lux. Retinal structure was assessed using spectral domain optical coherence tomography and retinal function was assessed using electroretinography. To investigate the mechanisms of flibanserin-mediated neuroprotection, gene expression, measured by RT-qPCR, was assessed following five days of daily 15 mg/kg flibanserin injections.
A five-day treatment regimen of 3 to 15 mg/kg of flibanserin significantly preserved outer retinal structure and function in a dose-dependent manner. Additionally, a single-day treatment regimen of 6 to 15 mg/kg of flibanserin still provided significant protection. The action of flibanserin was hindered by the 5-HT1A antagonist, WAY 100635, and was not effective in 5-HT1A knockout mice.
Intraperitoneal delivery of flibanserin in a light-induced retinopathy mouse model provides retinal neuroprotection. Mechanistic data suggests that this effect is mediated through 5-HT1A receptors and that flibanserin augments the expression of genes capable of reducing mitochondrial dysfunction and oxidative stress. Since flibanserin is already FDA-approved for other indications, the potential to repurpose this drug for treating retinal degenerations merits further investigation.
Inherited retinal dystrophies (IRDs), such as retinitis pigmentosa, are clinically and genetically diverse, and account for approximately 5% of the vision loss in the Western world. [
The neurotransmitter, serotonin (5-hydroxytryptamine or 5-HT), has been shown to modulate retinal processing, although the full extent of the role of serotonin in the retina remains unclear. [
Recent studies conducted by our lab and others suggest that certain serotonin receptor agonists and antagonists provide neuroprotection against light-induced retinopathy. [
Flibanserin, marketed as Addyi™ (Sprout Pharmaceuticals), was recently approved by the Food and Drug Administration (FDA) for the treatment of hypoactive sexual desire disorder (HSDD). [
The determination of retinal protection and cell survival mechanisms conferred by neuroprotective agents, such as flibanserin, is aided by evaluation in models with widely-described cell death mechanisms. The relatively synchronous and rapid progression of apoptosis in light-induced retinopathy has provided many insights into the mechanisms of cell death. [
All experiments and animal handling procedures were approved by and performed in compliance with the policies of the Institutional Animal Care and Use Committee at Oregon Health & Science University (IACUC Protocol Number IS03147) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Albino BALB/cJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). 5-HT1A-/- mice (5-HT1A knockout mice) were generously provided by Dr. René Hen (Columbia University, New York, NY). [
PCR analysis was performed using Quick-load Taq (Taq 2x Master Mix, New England Biolabs, Ipswich, MA) and the following primer sequences (Integrated DNA Technologies, San Diego, CA): 5-HT1A promoter
Flibanserin (Sigma-Aldrich, St. Louis, MO) was dissolved in Kollisolv PEG E 400 (PEG 400) (Sigma-Aldrich, St. Louis, MO), warmed to 60°C and diluted to 35% (v/v) in 0.9% saline (Hospira, Inc., Lake Forest, IL). The vehicle was 35% (v/v) PEG 400 in 0.9% saline. WAY 100635 was dissolved in 0.9% saline. The vehicle for this experiment was 0.9% saline.
Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. Mice were dosed with vehicle, 0.75, 1.5, 3.0, 6.0, 9.0 or 15 mg/kg flibanserin 48 hours, 24 hours and immediately prior to a one-hour bright light exposure, and 24 and 48 hours after light exposure. Naïve mice were not injected or exposed to bright light.
Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. A single dose of vehicle, 3, 6, 9 or 15 mg/kg of flibanserin was administered immediately before a one-hour bright light exposure in order to evaluate a shortened one-day treatment regimen. To assess 5-HT1A-mediated effects, 10 mg/kg WAY 100635, a selective 5-HT1A antagonist (Ki = 2),[
Four compact fluorescent lamps (42 watts, 6500K) were installed in a custom-built light box producing 10,000 lux of uniform light, and capable of holding 16 mice. [
Retinas were imaged as previously reported. [
SD-OCT scans were processed and segmented as previously reported. [
ERGs were performed as previously reported. [
Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. Mice were dosed with vehicle or 15 mg/kg flibanserin 48 hours, 24 hours and immediately prior to a one-hour bright light exposure, and 24 and 48 hours after light exposure. Retinas from vehicle-injected mice and flibanserin-injected mice were harvested 48 hours and 72 hours after light exposure. Total RNA was extracted from retinas using an RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was converted to cDNA using a Bio-Rad iScript cDNA synthesis kit (Hercules, CA). Primers were designed with the Integrated DNA Technologies Realtime PCR Tool (
Average outer retinal thickness (REC+), total retinal thickness (TR), a-wave amplitudes and b-wave amplitudes were calculated for each animal. Representative topographic distributions were an average of the REC+ thicknesses from the right eyes for all animals in each group, centered at the optic nerve. For scatterplot graphs, left and right eye data was combined for each animal and then averaged for each group. One-way analysis of variance (ANOVA) was used to compare REC+, TR and three different ERG measurements: bmax,rod at -2.14 log cd•s/m2, and amax,rod+cone and bmax,rod+cone at 3.55 log cd•s/m2 using Prism (Prism 6.0; GraphPad Software Inc., La Jolla, CA). Multiple t-tests were performed to compare the expression of individual genes in flibanserin- and vehicle-injected mice at 48- and 72-hours after light exposure. For all analyses, a
Bright light exposure resulted in thinning of the outer nuclear layer (ONL) and obscuration of the inner segment/outer segment junction (IS/OS) in retinas of vehicle-injected mice, and 0.75 and 1.5 mg/kg flibanserin-injected mice, as compared the normal morphology of naïve mice (
(
Nasal | Temporal | ||||
---|---|---|---|---|---|
Group | TR (μm) Mean ± SD | Rec+ (μm) Mean ± SD | TR (μm) Mean ± SD | Rec+ (μm) Mean ± SD | |
Vehicle | 10 | 125 ± 18 | 43 ± 19 | 117 ± 14 | 36 ± 14 |
Flibanserin, 0.75 mg/kg | 7 | 119 ± 8 | 37 ± 8 | 106 ± 4 | 25 ± 4 |
Flibanserin, 1.5 mg/kg | 7 | 132 ± 16 | 50 ± 16 | 113 ± 13 | 31 ± 11 |
Flibanserin, 3 mg/kg | 9 | 175 ± 20 | 94 ± 19 | 165 ± 19 | 82 ± 18 |
Flibanserin, 6 mg/kg | 7 | 179 ± 23 | 98 ± 23 | 168 ± 26 | 86 ± 26 |
Flibanserin, 9 mg/kg | 9 | 185 ± 14 | 104 ± 14 | 166 ± 21 | 85 ± 21 |
Flibanserin, 15 mg/kg | 9 | 196 ± 3 | 116 ± 2 | 191 ± 7 | 112 ± 7 |
Naive | 15 | 195 ± 2 | 115 ± 2 | 191 ± 2 | 112 ± 2 |
Vehicle | 7 | 136 ± 22 | 52 ± 21 | 119 ± 18 | 37 ± 17 |
Flibanserin, 3 mg/kg | 5 | 120 ± 12 | 39 ± 11 | 116 ± 21 | 36 ± 19 |
Flibanserin, 6 mg/kg | 6 | 194 ± 3 | 116 ± 2 | 192 ± 3 | 112 ± 2 |
Flibanserin, 9 mg/kg | 6 | 199 ± 2 | 120 ± 2 | 196 ± 2 | 118 ± 3 |
Flibanserin, 15 mg/kg | 7 | 197 ± 3 | 114 ± 2 | 194 ± 3 | 111 ± 2 |
Naive | 15 | 195 ± 2 | 115 ± 2 | 191 ± 2 | 112 ± 2 |
ERGs demonstrated severe attenuation of retinal function in vehicle-injected mice versus naïve mice, as observed in the representative ERG waveforms and scotopic a-wave and b-wave amplitudes (
(
Group | bmax,rod (μV) Mean ± SD | amax,rod+cone (μV) Mean ± SD | bmax,rod+cone (μV) Mean ± SD | |
---|---|---|---|---|
Vehicle | 10 | 262 ± 99 | 181 ± 71 | 365 ± 155 |
Flibanserin, 0.75 mg/kg | 7 | 146 ± 70 | 127 ± 50 | 301 ± 142 |
Flibanserin, 1.5 mg/kg | 7 | 203 ± 104 | 189 ± 91 | 386 ± 174 |
Flibanserin, 3 mg/kg | 9 | 398 ± 91 | 329 ± 80 | 666 ± 114 |
Flibanserin, 6 mg/kg | 7 | 468 ± 131 | 384 ± 123 | 807 ± 186 |
Flibanserin, 9 mg/kg | 9 | 525 ± 118 | 407 ± 71 | 785 ± 139 |
Flibanserin, 15 mg/kg | 9 | 556 ± 64 | 476 ± 86 | 945 ± 177 |
Naive | 15 | 606 ± 58 | 487 ± 68 | 973 ± 129 |
Vehicle | 7 | 251 ± 47 | 229 ± 51 | 424 ± 101 |
Flibanserin, 3 mg/kg | 5 | 226 ± 87 | 167 ± 88 | 393 ± 167 |
Flibanserin, 6 mg/kg | 5 | 579 ± 93 | 466 ± 118 | 917 ± 187 |
Flibanserin, 9 mg/kg | 6 | 464 ± 73 | 357 ± 110 | 668 ± 284 |
Flibanserin, 15 mg/kg | 7 | 513 ± 65 | 459 ± 53 | 934 ± 121 |
Naive | 15 | 606 ± 58 | 487 ± 68 | 973 ± 129 |
In order to ascertain whether flibanserin has the ability to act quickly and effectively, a one-day time course was evaluated. A single dose of 3, 6, 9 or 15 mg/kg flibanserin was injected immediately before light damage. Mice that received a 15, 9 or 6 mg/kg dose of flibanserin displayed complete morphological protection as assessed by OCT, and no statistical difference was found between their average REC+ thicknesses and those of naïve animals (
In regard to retinal function, all responses (bmax,rod, bmax,rod+cone, and amax,rod+cone) for the 6, 9 and 15 mg/kg flibanserin groups were statistically increased from the vehicle-treated group (
Because flibanserin is both a 5-HT1A agonist and a 5-HT2A antagonist, both receptors may play a role in flibanserin-mediated neuroprotection. However, literature suggests that flibanserin’s mechanism of action is primarily due to stimulation of 5-HT1A receptors. [
(
Potential cell survival mechanisms involved in flibanserin-mediated neuroprotection were assessed via reverse transcription-quantitative PCR (RT-qPCR); specifically, genes involved with the inhibition of apoptosis or reduction of reactive oxygen species were evaluated. At 48-hours post-light exposure, flibanserin treatment significantly increased gene expression of cAMP response element-binding protein (
This research demonstrates the novel finding that flibanserin, a dual serotonin receptor agonist and antagonist recently approved by the United States Food and Drug Administration for female HSDD, can completely protect the structural and functional integrity of albino BALB/c mouse retinas from light-induced retinopathy. Flibanserin’s neuroprotective effects began with a 3 mg/kg dose following a five-day time course and a 6 mg/kg dose following a one-day time course. A 15 mg/kg dose of flibanserin, following either time course, fully prevented the photoreceptor degeneration caused by bright light exposure.
Filbanserin demonstrated neuroprotection in a dose-dependent manner, consistent with the known pharmacokinetics of the drug. Borsini et al. showed that in the cortex, the 20% minimum receptor occupancy required for 5-HT1A receptor activation was reached after a single 1 mg/kg dose, but that a 10 mg/kg dose was required to reach the same level of receptor occupancy in the hippocampus and dorsal raphe. [
In humans, three daily-administrations of flibanserin are required for the drug to reach steady state plasma concentrations. [
Flibanserin has high affinity for the 5-HT1A receptor (Ki = 1 nM) and a lower affinity for the 5-HT2A receptor (Ki = 49nM), but lacks significant affinity for all other serotonergic and adrenergic receptors, allowing for the characterization of the neuroprotective targets, 5-HT1A and 5-HT2A, in combination. [
In HSDD, Flibanserin is thought to mediate its effect through modulation of serotonin, dopamine and norepinephrine levels in brain regions involved in activating dopaminergic reward and sexual cue integration. [
The signaling cascade involved in 5-HT1A-mediated neuroprotection has yet to be fully characterized, therefore we performed gene expression assays to elicit the mechanisms responsible for flibanserin-mediated neuroprotection. The cell death signaling implicated in light-induced retinopathy involves the activation of nitric oxide synthase (
Mitochondrial dysfunction often leads to oxidative stress, which occurs when reactive oxygen species capacity outweighs the antioxidant defense capacity. [
Flibanserin treatment also led to significant increases in
In summary, when light-damage occurs, there is an increase of intracellular calcium, disruption of mitochondrial function, and oxidative stress. [
Flibanserin was recently approved by the FDA and marketed as Addyi™ for the treatment of HSDD and its side effects are relatively benign in both men and women; the most frequent adverse events being dizziness, somnolence, nausea and fatigue. [
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